title: A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions.
abstract: Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1% and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3'-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.
authors.affiliation: National Center for Advancing Translational Sciences, National Institute of Health, 9800 Medical Center Drive, MSC: 3370, Bethesda, MD 20892-3370, USA.
authors.forename: Seameen J
authors.initials: SJ
authors.lastname: Dehdashti
authors.forename: Jennifer
authors.initials: J
authors.lastname: Abbott
authors.forename: Dac-Trung
authors.initials: DT
authors.lastname: Nguyen
authors.forename: John C
authors.initials: JC
authors.lastname: McKew
authors.forename: Peter R
authors.initials: PR
authors.lastname: Williamson
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D000935:Antifungal Agents / Q000008:administration & dosage*; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D003455:Cryptococcus neoformans / Q000187:drug effects* / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D000935:Antifungal Agents; D015415:Biomarkers; D000255:Adenosine Triphosphate